Cloning a disease gene on the basis of positional information is still an extremely labour-intensive and technically demanding process, made very much easier if additional information is available. One of the most helpful things to know is what genes are present in the region thought to contain the disease gene, permitting the researcher to test each candidate. (1°> Once an enormous undertaking, finding genes at a locus can now be done by anyone with access to the Internet, making positional candidate approaches to disease gene identification a very effective strategy. Genes, and more besides, are identified every day as part of the Human Genome Project and made publicly available at the websites of the participating laboratories (e.g. http://wwW.sao.g.e^.§c.lUk/ and htt2:l//wwwl.geoome..wi.mit.®dy/■) Procedures for the identification of disease genes are so inextricably tied up with the progress of the Human Genome Project that molecular genetic approaches to human health and illness cannot be discussed without considering the Project's aims and scope.
Ever since its inception there has been discussion as to whether resources should be focused on sequencing only coding DNA. The biological interest of the human genome lies primarily in the genes, which could be sequenced at a fraction of the cost and effort required to sequence the entire genome. Genes can be identified by isolating mRNA, using an enzyme (reverse transcriptase) to copy RNA into DNA, and then sequencing the DNA. Making RNA from many different tissues and from different developmental stages increases the chances of obtaining a representative collection of expressed sequences, and this project has been undertaken by a number of academic and industrial research groups/1,1,' Small fragments of DNA complementary to the mRNA (termed cDNA) are sequenced to generate a set of expressed sequence tags (ESTs) that are then placed on the existing physical and genetic maps of the human genome.(12) These advances mean that disease gene identification can be achieved by searching a database of ESTs and other sequence information within the region known to contain the disease locus. The size of the databases is enormous; at the time of writing publicly available databases include 1.7 million EST entries.
However, sequencing cDNA is now only a small portion of the effort that is being put into the Human Genome Project. A number of laboratories are devoted entirely to cloning, and then sequencing, human chromosomes, so that once a disease gene has been localized, the most efficient method to identify the abnormal transcript is to contact the appropriate laboratory. Much basic biology can then be done with a computer and a telephone. Indeed, bio-informatics is likely to take over from bench work as the major tool for disease gene identification.
Molecular techniques have been most successful in identifying genetic mutations in disorders with a simple genetic basis—one mutated gene segregating according to Mendelian genetics causing disease in a recessive or dominant fashion. Such single-gene mutations make a small contribution to psychiatric disorders, with the exception of syndromes associated with mental retardation. Can the same strategy work for common psychiatric disorders? There are three reasons to be cautious.
First, not all genetic conditions that have a simple genetic basis have yielded to positional cloning strategies. A good example is fascio-scapulo-humeral muscular dystrophy (FSHD) which has been mapped to the end of 4q and associated with the presence of an unusual series of repeats. Despite extensive characterization and sequencing of the region, no mutated genes have been found. The suspicion is that the repetitive region is involved in controlling a set of distant, as yet uncharacterized genes.(1, ,14> Similar complexities have beset attempts to clone genes implicated in some chromosomal deletion syndromes (see below). Thus our knowledge of gene regulation is not sufficiently advanced to permit us to recognize all mutations.
Second, it is known that the same phenotype can arise from mutations at different loci (genetic heterogeneity); conversely, mutations in a single gene can result in a set of different phenotypes (an allelic series). Perhaps the best example of an allelic series is the mutations in one of the fibroblast growth factor receptor genes (FGFR2) which are responsible for four different craniofacial syndromes. (15) No examples of allelic series are recorded (yet) in psychiatric genetics. However, genetic heterogeneity is already documented for Alzheimer's disease, which can be due to mutations in presenilin genes on chromosomes 1 and 14, and to mutations in the amyloid precursor protein gene on chromosome 21. (,1 l7 !8 and 19> Genetic heterogeneity is almost certainly an important feature of the genetic architecture of schizophrenia and manic-depressive psychosis. Both allelic series and genetic heterogeneity seriously complicate the task of associating genotypes with phenotypes, the basis of disease gene mapping and identification.
Third, the genetic contribution to many common psychiatric disorders may not be a mutation that disrupts gene expression but one that has a more subtle effect and is considerably harder to detect. Traits such as anxiety and depression are normally distributed quantitative traits for which the distinction between clinical disorder and normality is conventional. It is hard to believe that the molecular basis of allelic differences underlying quantitative traits is due to the type of mutation mentioned above. While the genetic basis for some extreme phenotypes might be due to the deletion of a gene or to an inactivating mutation, it is much more likely that extremes of traits such as anxiety and depression are due to subtle changes in gene expression, perhaps affecting developmental genes early in life and possibly arising from allelic differences in unexpressed regions of the genome that contain sequences controlling gene expression. Consequently it will be very hard to devise a way of proving that a candidate gene really does underlie the phenotype. There may be no discernible sequence differences or expression differences to identify the gene and even if there are, their presence does not prove that they are of aetiological significance.
While association studies can go some way to implicating a particular sequence, they can never be proof of a causal relation. A functional assay is needed—a way to alter the sequence and see whether that change results in an altered phenotype. Such experiments are currently only possible in animals and may be the only way to understand how genetic differences result in individual variation in susceptibility to common psychiatric disorders.
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